PROJECT SUMMARY The introduction of combination antiretroviral therapy (cART) in clinical practice has transformed the manage- ment of HIV-1 infection, achieving complete suppression of viral replication. However, cART does not target latent proviruses or the cells harboring them, and must be taken daily for life. The development of a cure for HIV- 1 infection would eliminate cART toxicities, and solve the economic and logistic burden of delivering cART to over 30 million patients worldwide, most of them in the developing world. Despite significant efforts in that direc- tion over the last decade, we are still far from an effective cure, underscoring the urgent need for new strategies. We argue that cART-free HIV-1 remission is more likely achieved by constraining latent proviruses into a deeper and permanent state of latency. Our long-term goal is to develop a ?block and lock? approach via epigenetic silencing of HIV-1 that is sequence-specific, safe, effective, and scalable throughout the world. This approach should aim at inducing irreversible epigenetic inactivation of HIV-1, in a manner similar to permanent silencing of tissue-specific genes and endogenous retroelements within our human genomes. Our overall objective is to achieve robust, durable, sequence-specific silencing of latent HIV-1 proviruses that leads to their permanent transcriptional inactivation. Our central hypothesis is that exogenous expression of a naturally occurring HIV-1 antisense transcript (Ast) will lead to sequence-specific silencing of latent HIV-1. We have reported that the Ast RNA naturally impairs HIV-1 expression by binding to homologous DNA sequences in the proviral 5?LTR through base pairing and by recruiting the Polycomb Repressor Complex 2 (PRC2), which introduces the repressive epigenetic mark, H3K27me3 into the surrounding chromatin, turning off HIV-1 expres- sion. In addition, our recent studies show that the Ast RNA binds additional repressive epigenetic and transcrip- tional factors, suggesting that this transcript orchestrates multiple mechanisms of HIV-1 silencing. We propose to achieve our goal through three Specific Aims (SA). SA1 will investigate modifications of the Ast RNA, and additional epigenetic repressors that Ast recruits to the HIV-1 5?LTR. These studies will lead to devel- oping Ast derivatives with improved potency. SA2 will evaluate the ability of the Ast RNA and its new derivatives to induce sequence-specific silencing of HIV-1 in three in vitro and ex vivo biologically relevant primary cell models. These studies represent the first level of testing for Ast and its derivatives, providing helpful feedback to SA1 studies and selecting Ast derivatives for further testing. SA3 will evaluate the ability of Ast RNA and its derivatives selected in SA2 to induce HIV-1 silencing in an in vivo humanized mouse model reconstituted with CD4+ T cells from HIV-1 patients. These studies are a second and more complex level of testing for Ast RNA. Completion of these studies will provide proof-of-concept that could propel the development of the Ast RNA into a novel, potent and sequence-specific therapeutic for HIV-1 cure. While most silencing strategies have a broad impact on gene expression that is likely accompanied by toxicity, the Ast RNA will allow to launch a targeted, sequence-specific strike against latent proviruses, avoiding significant toxic off-target effects.